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ASTM D5259-92(2006)

(Test Method)

Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure

Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure

SIGNIFICANCE AND USE
The enterococci are indicators of the bacteriological quality for potable water, shellfish growing waters, ambient, and recreational waters. A direct relationship between swimming, associated gastroenteritis, and enterococci has been established through epidemiological studies and marine and fresh water bathing beaches. These studies have led to the development of criteria that can be used to establish bathing water standards based on established health-water quality relationships.
Since small or large volumes of water or dilutions thereof, can be analyzed by the membrane filter technique, a wide range of levels of enterococci in water can be enumerated and detected.
SCOPE
1.1 This test method covers a membrane filter (MF) procedure for the detection and enumeration of the enterococci bacteria in water. The enterococci, which include Entero-coccus faecalis (E. faecalis),  E. faecium, and their varieties are commonly found in the feces of humans and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution, enterococci in water are an indication of fecal pollution and the possible presence of enteric pathogens. These bacteria are found in water and wastewater in a wide range of densities. The detection limit is one colony forming unit (CFU)/volume filtered.
1.2 This test method has been used successfully with temperate fresh and marine ambient waters, and wastewaters. It is the user's responsibility to ensure the validity of this test method for waters of untested types.
1.3 The values stated in SI units are to be regarded as the standard.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. For specific hazard statements, see Section .

General Information

Status
Historical
Publication Date
30-Jun-2006
ICS
13.060.70 - Examination of biological properties of water
Technical Committee
D19 - Water
Drafting Committee
D19.24 - Water Microbiology
Current Stage
Ref Project

Relations

Replaces
ASTM D5259-92(2000) - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
Effective Date
01-Jul-2006
Replaced By
ASTM D5259-92(2012) - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
Effective Date
01-Jun-2012
Referred By
ASTM E1891-21 - Standard Guide for Determination of a Survival Curve for Antimicrobial Agents Against Selected Microorganisms and Calculation of a D-Value and Concentration Coefficient
Effective Date
01-Jul-2006
Referred By
ASTM D6974-20 - Standard Practice for Enumeration of Viable Bacteria and Fungi in Liquid Fuels—Filtration and Culture Procedures
Effective Date
01-Jul-2006
Referred By
ASTM E3152-23 - Standard Guide for Standard Test Methods and Practices Available for Determining Antifungal Activity on Natural or Synthetic Substrates Treated with Antimicrobial Agents
Effective Date
01-Jul-2006
Referred By
ASTM D5465-16(2020) - Standard Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
Effective Date
01-Jul-2006

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ASTM D5259-92(2006) - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter ProcedureASTM D5259-92(2006) - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
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ASTM D5259-92(2006) - Standard Test Method for Isolation and Enumeration of Enterococci from Water by the Membrane Filter Procedure
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Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: D5259 − 92 (Reapproved 2006)
StandardTest Method for
Isolation and Enumeration of Enterococci from Water by the
1
Membrane Filter Procedure
This standard is issued under the fixed designation D5259; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope for Colony Counting Methods in Microbiology (With-
3
drawn 2000)
1.1 This test method covers a membrane filter (MF) proce-
dure for the detection and enumeration of the enterococci
3. Terminology
bacteria in water. The enterococci, which include Entero-
3.1 Definitions—For definitions of terms used in this test
coccus faecalis (E. faecalis), E. faecium, and their varieties are
method, refer to Terminology D1129.
commonly found in the feces of humans and other warm-
blooded animals.Although some strains are ubiquitous and not
3.2 Definitions of Terms Specific to This Standard:
relatedtofecalpollution,enterococciinwaterareanindication
3.2.1 Enterococcus—In this test method, Enterococcus spe-
of fecal pollution and the possible presence of enteric patho-
ciesarethosebacteriathatproduceredtomarooncolonieswith
gens. These bacteria are found in water and wastewater in a
black or reddish-brown precipitate on underside, after incuba-
wide range of densities. The detection limit is one colony
tion on mE agar and subsequent transfer to EIA medium.
forming unit (CFU)/volume filtered.
Enterococciinclude E. faecalis, E. faecium, E. avium,andtheir
variants.
1.2 This test method has been used successfully with
temperatefreshandmarineambientwaters,andwastewaters.It
4. Summary of Test Method
is the user’s responsibility to ensure the validity of this test
4.1 The procedure given in this test method provides a
method for waters of untested types.
direct count of bacteria in water based on the development of
1.3 The values stated in SI units are to be regarded as the 4
coloniesonthesurfaceofthemembranefilter. Awatersample
standard.
is filtered through the membrane that retains the bacteria.
1.4 This standard does not purport to address all of the Following filtration, the membrane containing the bacterial
safety concerns, if any, associated with its use. It is the cells is placed on a selective, medium, mE agar, and incubated
responsibility of the user of this standard to establish appro- for 48 h at 41°C, then transferred to EIAagar and held at 41°C
priate safety and health practices and determine the applica- for20min.Enterococcidevelopasredtomarooncolonieswith
bility of regulatory limitations prior to use. For specific hazard black or reddish-brown precipitate on the underside of the
statements, see Section 9. filter.
5. Significance and Use
2. Referenced Documents
2
5.1 The enterococci are indicators of the bacteriological
2.1 ASTM Standards:
quality for potable water, shellfish growing waters, ambient,
D1129 Terminology Relating to Water
and recreational waters. A direct relationship between
D1193 Specification for Reagent Water
swimming,associatedgastroenteritis,andenterococcihasbeen
D3370 Practices for Sampling Water from Closed Conduits
established through epidemiological studies and marine and
D3870 PracticeforEstablishingPerformanceCharacteristics
fresh water bathing beaches. These studies have led to the
development of criteria that can be used to establish bathing
water standards based on established health-water quality
1
This test method is under the jurisdiction of ASTM Committee D19 on Water
relationships.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved July 1, 2006. Published July 2006. Originally approved
in 1992. Last previous edition approved in 2000 as D5259 – 92 (2000). DOI:
3
10.1520/D5259-92R06. The last approved version of this historical standard is referenced on
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or www.astm.org.
4
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Cabelli,V. J., Dufour,A. P., Levin, M.A., McCabe, L. J., and Haberman, P.W.,
Standards volume information, refer to the standard’s Document Summary page on “RelationshipofMicrobialIndicatorstoHealthEffectsatMarineBathingBeaches,”
the ASTM website. American Journal of Public Health, 69, 1979, pp. 690–696.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
1

---------------------- Page: 1 ----------------------
D5259 − 92 (Reapproved 2006)
5.2 Since small or large volumes of water or dilutions 7.14 Bottles, milk dilution, borosilicate glass, screw-cap
thereof, can be analyzed by the membrane filter te
...

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SIGNIFICANCE AND USE
5.1 The enterococci are indicators of the bacteriological quality for potable water, shellfish growing waters, ambient, and recreational waters. A direct relationship between swimming, associated gastroenteritis, and enterococci has been established through epidemiological studies and marine and fresh water bathing beaches. These studies have led to the development of criteria that can be used to establish bathing water standards based on established health-water quality relationships.  
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SIGNIFICANCE AND USE
5.1 This test method is an alternative for the silt density index (SDI) method (Test Method D4189) with the aim to overcome inaccuracies related to a nonlinear relation with the fouling potential due to particulate matter concentration, absence of temperature correction, support pad, and time.  
5.2 The MFI-0.45 can serve as a useful indication of the quantity of particulate matter.  
5.3 The MFI-0.45 can be used to determine effectiveness of various processes such as filtration or clarification used to remove particulate matter.  
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1.2 This test method is not applicable for reagent-grade water Types I, II, and III of Specification D1193 or effluents from most reverse osmosis and ultra-filtration systems.  
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ASTM D5864-23(Test Method)
Standard Test Method for Determining Aerobic Aquatic Biodegradation of Lubricants or Their Components

SIGNIFICANCE AND USE
5.1 Results from the test method suggest, within the confines of a controlled laboratory setting, the degree of aerobic aquatic biodegradation of a lubricant or components of a lubricant by measuring the evolved carbon dioxide upon exposure of the test material to an inoculum. The plateau level of CO2 evolution in this test method will suggest the degree of biodegradability of the lubricant. Test substances that achieve a high degree of biodegradation in this test may be assumed to easily biodegrade in many aerobic aquatic environments.  
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This guide covers procedures for obtaining laboratory data concerning the adverse effects of aqueous ambient samples and effluents on certain species of freshwater and saltwater fishes, macroinvertebrates, and amphibians, during a short-term exposure, depending on the species, using the static, renewal, and flow-through techniques. These procedures will probably be useful for conducting acute toxicity tests on aqueous effluents with many other aquatic species, although modifications might be necessary. Static tests might not be applicable to effluents that have a high oxygen demand, or contain materials that (1) are highly volatile, (2) are rapidly biologically or chemically transformed in aqueous solutions, or (3) are removed from test solutions in substantial quantities by the test chambers or organisms during the test. Results of acute toxicity tests should usually be reported in terms of a median lethal concentration (LC50) or median effective concentration (EC50). An acute toxicity test does not provide information about whether delayed effects will occur. Specified requirements involving the following are detailed: (1) hazards; (2) apparatus: facilities, special requirements, construction materials, metering system, test chambers, cleaning, and acceptability; (3) dilution water requirements, source, treatment, and characterization; (4) effluent sampling point, collection, preservation, treatment, and test concentrations; (5) test organism species, age, source, care and handling, feeding, disease treatment, holding, acclimation, and quality; (6) procedure: experimental design, dissolved oxygen, temperature, loading, beginning the test, feeding, duration of test, biological data, and other measurements; (7) analytical methodology; (8) acceptability of test; (9) calculation of results; and (1) report of results.
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5.1 The ecological importance of bivalves, their wide geographic distribution, ease of handling in the laboratory and the field, and their ability to filter and ingest large volumes of water and sediment particles make them appropriate species for conducting field bioassays to assess bioaccumulation potential and associated biological effects. The test procedures in this guide are intended to provide guidance for conducting controlled experiments with caged bivalves under “natural,” site-specific conditions. It is important to acknowledge that a number of “natural” factors can affect bivalve growth and the accumulation of chemicals in their tissues (Section 6, Interferences). This field bioassay can also be conducted in conjunction with laboratory bioassays to help answer questions raised in the field exposures. The field exposures can also be used to validate the results of laboratory bioassays.  
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5.1 While federal criteria and state standards exist that define acute and chronic “safe” levels in the water column, effects levels in the sediment are poorly defined and may be dependent upon numerous modifying factors. Even where USEPA recommended Water Quality Criteria (WQC, (49)) are not exceeded by water-borne concentrations, organisms that live in or near the sediment may still be adversely affected (50). Therefore, simply measuring the concentration of a chemical in the sediment or in the water is often insufficient to evaluate its actual environmental toxicity. Concentrations of contaminants in sediment may be much higher than concentrations in overlying water; this is especially true of hydrophobic organic compounds as well as inorganic ions that have a strong affinity for organic ligands and negatively-charged surfaces. Higher chemical concentrations in sediment do not, however, always translate to greater toxicity or bioaccumulation (51), although research also suggests that amending sediment with organic matter actually increases the bioaccumulation of contaminant particles (52, 53). Other factors that can potentially influence sediment bioaccumulation and toxicity include pH mineralogical composition, acid-volatile sulfide (AVS) grain size, and temperature (54-56). Laboratory toxicity tests provide a direct and effective way to evaluate the impacts of sediment contamination on environmental receptors while providing empirical consideration of all of the physical, chemical and biological parameters that may influence toxicity.  
5.2 Amphibians are often a major ecosystem component of wetlands around the world, however limited data are available regarding the effects of sediment-bound contaminants to amphibians (39, 41, 43, 55, 57, 58). Laboratory studies such as the procedure described in this standard are one means of directly assessing sediment toxicity to amphibians in order to evaluate potential ecological risks in wetlands.  
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1.1 This standard covers procedures for obtaining laboratory data concerning the toxicity of test material (for example, sediment or hydric soil (that is, a soil that is saturated, flooded, or ponded long enough during the growing season to develop anaerobic (oxygen-lacking) conditions that favor the growth and regeneration of hydrophytic vegetation)) to amphibians. This test procedure uses larvae of the northern leopard frog (Lithobates pipiens). Other anuran species (for example, the green frog (Lithobates clamitans), the wood frog (Lithobates sylvatica), the American toad (Bufo americanus)) may be used if sufficient data on handling, feeding, and sensitivity are available. Test material may be sediments or hydric soil collected from the field or spiked with compounds in the laboratory.  
1.2 The test procedure describes a 10-d whole sediment toxicity test with an assessment of mortality and selected sublethal endpoints (that is, body width, body length). The toxicity tests are conducted in 300 to 500-mL chambers containing 100 mL of sediment and 175 mL of overlying water. Overlying water is renewed daily and larval amphibians are fed during the toxicity test once they reach Gosner stage 25 (operculum closure over gills). The test procedure is designed to assess freshwater sediments, however, R. pipiens can tolerate mildly saline water (not exceeding about 2500 mg Cl-/L, equivalent to a salinity of about 4.1 when Na+ is the cation) in 10-d tests, although such tests should always include a concurrent freshwater control. Alternative test durations and sublethal endpoints may be considered based on site-specific needs. Statistical evaluations are conducted to determine whether test materials are significantly more toxic than the laboratory control sediment or a field-collected reference sample(s).  
1.3 Where appropriate, this standard has been designed to be consistent with previously developed methods for assessing s...

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Standard Test Method for Retention Characteristics of 0.40 to 0.45 μm Membrane Filters Used in Routine Filtration Procedures for the Evaluation of Microbiological Water Quality

SIGNIFICANCE AND USE
5.1 Microbiological water testing procedures using membrane filtration are based on the premise that all bacteria within a specific size range will be retained by the membrane filter used. If the membrane filter does not retain these bacteria, false negative results or lowered density estimates may occur that could have serious repercussions due to the presence of unrecognized potential health hazards in the water being tested, especially in drinking water.  
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SIGNIFICANCE AND USE
5.1 The test procedure covered in this guide is not intended to simulate exactly the exposure of benthic polychaetes to chemicals under natural conditions, but rather to provide a conveniently rapid, standard toxicity test procedure yielding a reasonably sensitive indication of the toxicity of materials in marine and estuarine sediments.  
5.2 The protection of a community of organisms requires averting detrimental contaminant-related effects on the number and health of individuals and species within that population. Sediment toxicity tests provide information on the toxicity of test materials in sediments. Theoretically, projection of the most sensitive species within a community will protect the community as a whole.  
5.3 Polychaetes are an important component of the benthic community. They are preyed upon by many species of fish, birds, and larger invertebrate species, and they are predators of smaller invertebrates, larval stages of invertebrates, and, in some cases, algae, as well as organic material associated with sediment. Polychaetes are sensitive to both organic and inorganic chemicals (1, 2).5 The ecological importance of polychaetes, their wide geographical distribution and ability to be cultured in the laboratory, and sensitivity to chemicals, make them appropriate toxicity test organisms.  
5.4 An acute or 10-day toxicity test is conducted to obtain information concerning the immediate effects to a test material on a test organism under specified experimental conditions for a short period of time. An acute toxicity test does not necessarily provide information concerning whether delayed effects will occur, although a post-exposure observation period, with appropriate feeding, if necessary, could provide such information.  
5.5 The results of acute sediment toxicity tests can be used to predict acute effects likely to occur on aquatic organisms in field situations as a result of exposure under comparable conditions, except that (1) motile organisms...
SCOPE
1.1 This guide covers procedures for obtaining laboratory data concerning the adverse effects of potentially contaminated sediment, or of a test material added experimentally to contaminated or uncontaminated sediment, on marine or estuarine infaunal polychaetes during 10-day or 20 to 28-day exposures. These procedures are useful for testing the effects of various geochemical characteristics of sediments on marine and estuarine polychaetes and could be used to assess sediment toxicity to other infaunal taxa, although modifications of the procedures appropriate to the test species might be necessary. Procedures for the 10-day static test are described for Neanthes arenaceodentata and Alitta virens 2 (formerly Nereis virens and Neanthes virens) and for the 20 to 28-day static-renewal sediment toxicity for  N. arenaceodentata.  
1.2 Modifications of these procedures might be appropriate for other sediment toxicity test procedures, such as flow-through or partial life-cycle tests. The methods outlined in this guide should also be useful for conducting sediment toxicity tests with other aquatic taxa, although modifications might be necessary. Other test organisms might include other species of polychaetes, crustaceans, and bivalves.  
1.3 Other modifications of these procedures might be appropriate for special needs or circumstances. Although using appropriate procedures is more important than following prescribed procedures, the results of tests conducted using unusual procedures are not likely to be comparable to those of many other tests. Comparisons of the results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting sediment tests with infaunal organisms.  
1.4 These procedures are applicable to sediments contaminated with most chemicals, either individually or in formulations, commercial products, and known or unknown...

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ASTM
ASTM D3978-21a(Practice)
Standard Practice for Algal Growth Potential Testing with Pseudokirchneriella subcapitata

SIGNIFICANCE AND USE
5.1 The significance of measuring algal growth potential in water samples is that differentiation can be made between the nutrients of a sample determined by chemical analysis and the nutrients that are actually available for algal growth. The addition of nutrients (usually nitrogen and phosphorus singly or in combination) to the sample can give an indication of which nutrient(s) is (are) limiting for algal growth  (1,10,11,12,13,14).
SCOPE
1.1 This practice measures, by Pseudokirchnereilla subcapitata growth response, the biological availability of nutrients, as contrasted with chemical analysis of the components of the sample. This practice is useful for assessing the impact of nutrients, and changes in their loading, upon freshwater algal productivity. Other laboratory or indigenous algae can be used with this practice. However, Pseudokirchnereilla subcapitata must be cultured as a reference alga along with the alternative algal species.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. For a specific precautionary statement, see Section 16.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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